T cell-independent eradication of experimental glioma by intravenous TLR7/8-agonist-loaded nanoparticles

Glioblastoma, the most common and aggressive primary brain tumor type, is considered an immunologically “cold” tumor with sparse infiltration by adaptive immune cells. Immunosuppressive tumor-associated myeloid cells are drivers of tumor progression. Therefore, targeting and reprogramming intratumoral myeloid cells is an appealing therapeutic strategy. Here, we investigate a β-cyclodextrin nanoparticle (CDNP) formulation encapsulating the Toll-like receptor 7 and 8 (TLR7/8) agonist R848 (CDNP-R848) to reprogram myeloid cells in the glioma microenvironment. We show that intravenous monotherapy with CDNP-R848 induces regression of established syngeneic experimental glioma, resulting in increased survival rates compared with unloaded CDNP controls. Mechanistically, CDNP-R848 treatment reshapes the immunosuppressive tumor microenvironment and orchestrates tumor clearing by pro-inflammatory tumor-associated myeloid cells, independently of T cells and NK cells. Using serial magnetic resonance imaging, we identify a radiomic signature in response to CDNP-R848 treatment and ultrasmall superparamagnetic iron oxide (USPIO) imaging reveals that immunosuppressive macrophage recruitment is reduced by CDNP-R848. In conclusion, CDNP-R848 induces tumor regression in experimental glioma by targeting blood-borne macrophages without requiring adaptive immunity.

All data sets generated or analysed during this study are included in this published article (and its supplementary information files). Custom written code is publicly available: The CNN for radiomic response prediction is available at https://github.com/NeuroAI-HD/HD-GLIOMOUSE. The radiomic brain extraction was performed with MITK, https://phabricator.mitk.org/w/mitk/changelog/release-v2022.10. n/a n/a n/a n/a Sample size was calculated with the help of a biostatistician using R version 4.1.0 and as previously descrived (Aslan et al. Nat Comm., 2020). Assumptions for power analysis were as follows: alpha error: 5%; beta error: 20%. Values for standard deviation and differences between experimental groups were based on previous experiments. (whenever a similar data type was available). In all other cases a pilot group size was used.
In case animals had to be sacrificed prior to the pre-defined endpoint (due to weight loss or other termination criteria), they were excluded from any downstream analysis.
Key experiments (response to CDNP-R848, tumor microenvironment phenotyping by flow cytometry) were all performed at least twice and data from one representative experiment or pooled data are shown in this manuscript as indicated in the figure captions. Replication of experiments were successfull and showed comparable results. All other experiments were performed once with biological replicates (as specified in figure legend).
Mice were matched into the groups according to tumor size (measured by MRI) at the time of treatment start so ensure equal tumor volumes in the different treatmet arms. Tumor volumes at baseline were measured in a standardized manner before randomization was performed Intracranial tumor and in vitro BMDM experiments were performed in a blinded manner so the experimentation and analysis were blinded for treatment arm (MRI tumor volume assessment, flow cytometric analyses, qRT-PCR, histology). Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots. Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information. Cell lines were tesed negative for mycoplasma contamination regularly and before in vivo use. Highthroughput Multiplex Cell contamination (McCT), Schmitt M. et al. 2009 no commonly misidentified lines from the ICLAC register were used in this study C57BL/6J or C57BL/6N wild-type (WT) mice were purchased from Janvier Labs. Female age-matched mice were used for in vivo experiments. All mice were 7-12 weeks of age at use. Mice were kept under SPF conditions at the animal facility of the DKFZ Heidelberg.
n/a female mice were used in this study. It is expected that animal gender does not influence the results n/a All animal protocols were performed in compliance with the laboratory animal research guidelines and were approved by the governmental authorities (animal protocols: G27-17 and G35-22, regional administrative authority, Regierungspräsidium Karlsruhe, Germany) Murine Gl261-containing brain hemispheres were excised, washed in HBSS (Sigma-Aldrich) and cut into small pieces before tissue disruption in HBSS supplemented with 50 µg/ml Liberase D for 0,5 h under slow rotation at 37°C. Dispersed tissue was mashed through a 100µm and 70 !m cell strainer and lymphocytes. For Gl261 sampleas, myelin removal was performed by percoll density gradient as described in online methods. Murine splenocytes were isolated by homogenization using a cell strainer and ACK lysis. In some cases, as described in the online methods, cells were treated with Brefeldin A to prevent secretion of cytokines, chemokines, and other secretory proteins before analysis. Generation of BMDM cultures was performed according to previously established protocols. Briefly, bone marrow cells were flushed from the tibia and femurs of C57BL/6N wild-type mice (8-10 weeks of age) using ice-cold Hanks' Balanced Salt Solution (HBSS) and filtered through a 70 m cell strainer and plated at a density of 3.5 x 105 cells/ml. Cells were differentiated for one week using RPMI medium supplemented with 10 ng/ml M-CSF (M9170, Sigma-Aldrich), 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin (Gibco). As contrast agent 0.2 mmol/kg Dotarem (Guerbet) was administered i.v. to assess BBB integrity with T1-w. MGE T2* relaxometry was used for macrophage tracking using the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle ferumoxytol (Feraheme; AMAG Pharmaceuticals Inc.). Imaging was performed before and 24 hours after ferumoxytol (dose of 30mg/ kg). whole brain scan Single shell 2D EPI-DTI was acquired with 30 diffusion gradient directions (DTI Spinecho EPI, 4 Segments, Double Sampling, FOV 12mm x 15mm, Matrix 96 x 128, Partial Fourier Factor 1.5, 17 Slices, Slice Thickness 0.7mm, TE 20ms, TR 3400ms, Fat Suppression, FOV Saturation, Bandwith 333333Hz, Diffusion encoding: Gradient duration 3 ms, Gradient Separation 9 ms, Bvalue 1500 s/mm²). Cardiac-gating was not applied.
MR Images were exported as DICOM files and preprocessed in nordicICE Software (v4.2.0). For the quantification of MRI diffusion data, FA (fractional anisotropy) of the tumor core was segmented manually. The ratio of normal brain tissue and the tumor diffusion FA was calculated.
MRI data were neither normalized for tumor volumetry performed on T2w-images nor for analysis of FA-values in the tumor ROI. The calculation of T2* relaxation times included noise filtering. Sigma was calculated for the whole dataset and voxels with a deviation larger than 4 sigma were removed. Radiomics analysis included brain extraction and Z-score normalization of T2w-datasets.
Data were not normalized to a standard space.
The calculation of T2* relaxation times included noise filtering. Sigma was calculated for the whole dataset and voxels with a deviation larger than 4 sigma were removed. All other data were not processed for noise or artifact removal.
MR images were exported as DICOM files and were visualized in OsiriX Imaging software (version 4.12; Pixmeo) and FIJI (FIJI ImageJ, Version 1.52). For the quantification of MRI data, tumor volumes were segmented semi-automatically using AMIRA (FEI). Volumes were exported to Microsoft Excel. No censoring was performed.
Machine-learning-Gradient boosting in R version 4.0.3 (R Foundation for Statistical Computing, Vienna, Austria) using the caret library. Tuning parameters (boosting iterations, max tree depth, shrinkage and min. terminal node size) were automatically optimized via resampling procedures.